The transfection efficiency of mir21 and lipofection protocol. Transfecting sirna using lipofectamine rnaimax pin. Cas9 nuclease may be used in vivo to create targeted genome modifications. Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2. Transfection and genome engineering fisher scientific. Comparison of hsv, adenovirus, aav, retrovirus and lentivirus for gene delivery. Lipofectamine 3000 is a transfection reagent that was developed to improve transfection efficiency in many difficult to. To achieve the maximal rnai activity and lowest cytotoxicity, optimization of. Reverse transfection an overview sciencedirect topics. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a. Our data provide a broadranging comparison of transfection methods for.
In this study, we used three transfection reagents lipofectamine rnaimax, oligofectamine and lipofectamine 2000 to deliver sirna into human embryonic stem hes cells and compared the silencing efficiency of enhanced green fluorescent protein transgene and oct4, a key regulator of pluripotency. Lipofectamine rnaimax reagent protocol 20 typical rnaimax transfection procedure transfect cells according to the following table. Plate cells so they will be 6080% confluent at the time of transfection. Tube a contains optimem media and rnaimax reagent and tube b contains optimem media and sirna. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. May 21, 2018 to assess the efficiency of sirna uptake by sw1463 3d tumour spheroids we used a fluorescent cy3conjugated nontargeting control sirna and formed complexes with lipofectamine rnaimax in either. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. No other sirna specific transfection reagent provides such easy and efficient sirna delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hardtotransfect cell types. It is used to increase the transfection efficiency of rna. According to our results, lipofectamine 3000, fugene and rnaimax showed high transfection efficacy, however, rnaimax may be a better option for majority of cells when lower toxicity is desired. I am using the standard protocol for lipofectamine rnaimax plate cells to 6080% confluency, dilute 9l lipofectamine in 150l optimem, dilute 30 pmol sirna in 150l optimem. Lipofectamine 2000 transfection protocol 10cm plates reagents.
To transfect cells with sirna, follow the protocol as described for dna but do not add. You can make a master mix of the rnaimax and optimem and add this directly. Rapid and highly efficient mammalian cell engineering via. Comparative transfection efficiency of jetprime versus its main competitor. Amounts of reagent and sirna can be scaled up or down depending on the number of cells that will be transfected. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Invitrogen lipofectamine 2000 transfection reagent fisher. Mix lipofectamine rnaimax gently before use, then add 1 l lipofectamine rnaimax to each. The intracellular trafficking mechanism of lipofectamine. It takes nearly 120 barrels of crude oil equivalents and 150 mwh of power annually to make the 9,000 kg of eps coolers needed to ship these reagents1,2. Expression of lectinfc fusion protein in the culture supernatant was.
Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent and suspension cell lines. Lipofectamine rnaimax reagent thermo fisher scientific kr. Concentration of dna for stable transfection with lipofectamine 2000 and the per transfection and transduction. By shipping these reagents at ambient temperatures. Invitrogen lipofectamine rnaimax transfection reagent offers an advanced, efficient solution for sirna delivery.
Molecules free fulltext systematic screening of commonly. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. Dilute 6 pmol rnai duplex in 100 l optimem i medium without serum in the well of the tissue culture plate. Lipofectamine rnaimax rnaimax, a new cationic lipofection reagent, has shown. Lipofectamine rnaimax transfection reagent from thermo fisher.
Hek293t cells were transfected with prccmvlecfc using lipofectamine 2000, and culture supernatants were collected. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Dna complexes were added to cells that had already been allowed to. For the neon 24well optimization protocol, 24 g cas9 protein and 6 g. Rnaimax reverse transfections lipofectamine thermo fisher. Cloned genes can be transfected into cells for biochemical characterization. Invitrogen lipofectamine rnaimax transfection reagent.
After 24 hours, transfection with lipofectamine 2000 and rnaimax resulted in. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. The transfection is designed for one rna amount combined with one amount of lipofectamine rnaimax. Establishment of lipofection protocol for efficient mir21. Transfection efficiency and cell toxicityviability, reagent dose response a dose response from. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Optimized delivery of sirna into 3d tumor spheroid cultures.
The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Incubate 5 min at room temp then mix lipofectamine and sirna dilutions. So i asked one of my lab mates and he told me to use just 1ul lipofectamine for 20pmol of sirna thats the protocol specificationsbut. I use 24well plates for transfections in triplicates. Maintain p19 cells undifferentiated in mem with 10% serum 7. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Invitrogen lipofectamine 3000 invitrogen lipofectamine 3000 transfection reagent 1. Add 6l of lipofectamine2000 from biostores and 100 l of. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.
See the engen sgrna synthesis kit manual for further details. Day 0 day 1 day 24 cells lipofectamine 2000 reagent dna. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. What is the major character of lipofectamine 2000 as a transfection reagent. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates.
Rnaimax reverse transfections lipofectamine thermo. Invitrogen lipofectamine rnaimax transfection reagent 0. Advanced transfection with lipofectamine 2000 reagent. What is the major character of lipofectamine 2000 as a. Lipofectamine 3000 and lipofectamine rnaimax served as controls. E2311, promega, sydney, australia, lipofectamine 2000 reagent. Optimized delivery of sirna into 3d tumor spheroid. There are several ways in which to introduce cas9guide rna complexes into cells. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine 2000 is a highperformance transfection reagent for gene expression and gene silencing. Whats the difference between lipofectine and lipofectamine. Rnabased, transient modulation of gene expression in human. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. In this study, we used three transfection reagents lipofectamine rnaimax, oligofectamine and lipofectamine 2000 to deliver sirna into human embryonic stem hes cells and compared the. Optimized conditions for successful transfection of human. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. After the 10 min incubation, lipofectamine and sirna solution are combined, and the total of. Dilute lipofectamine in serum and antibioticfree media in a different 15ml tube. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna.
Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. I did get the knockdown to work when using my sirna with lipofectamine 2000, however. This protocol is optimized for transfection in a 6well in vitro culture plate format to perform. This protocol is optimized for transfection in a 6well in vitro culture plate format to perform transfection in different cell culture formats, refer to table2 and invitrogen lipofectamine rnaimax protocol. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Transfection of cas9 rnp ribonucleoprotein into adherent cells. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. For each well to be transfected, prepare rnai duplexlipofectamine rnaimax complexes as follows. Lipofectamine 2000 and lipofectamine rnaimax transfection.
Can anybody give me exact protocol for sirna transfection using. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine rnaimax reagent thermo fisher scientific us. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine rnaimax transfection reagent from thermo.
Invitrogen lipofectamine 2000 transfection reagent. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Add tube b to tube a and incubate at room temperature for five minutes. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid. Works effectively with all common cell lines as well as with many challenging ones, and can. Lipofectaminez 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium. Optimizing transfections to obtain the highest transfection efficiency and low nonspecific. Lipofectaminea rnaimax reagent thermo fisher scientific. Kit miltenyi biotec according to the manufacturers instructions. Transfection of cas9 rnp ribonucleoprotein into adherent. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1, which is in agreement with the recent finding that rnaimax performed better than lipofectamine 2000 for delivery of cas9 rnps with low cell toxicity zuris et al. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Because the cells and sirnalipid complexes are prepared on the same day, we. No 20 is optimized for use with lipofectamine 2000, and is not recommended for lipofectamine rnaimax.
What is critical for the efficiency and specificity of an rnai experiment is not the sirna concentration in the medium but. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectamine rnaimax reagent protocol thermo fisher scientific. Rnaimax transfection reagent 778030, invitrogen, waltham. The transfection is designed for one rna amount combined.
However, significant toxicity from lipofectamine 2000 was also reported in. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. Qc specifications back to table of content lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Systematic screening of commonly used commercial transfection. Lipofectamine rnaimax transfection reagent thermofisher.
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